The overall goal of this laboratory is to achieve long-term expression of a desired gene in vivo in a significant percentage of keratinocytes by identifying and introducing genes into keratinocyte stem cells (KSC). Skin gene therapy is an area of great potential. There are several potential applications for skin gene therapy that require either transient or long-term expression of a desired gene. One possible application is the correction of inherited skin disorders due to single gene mutations. Included in this category are a number of forms of epidermolysis bullosa (inherited blistering diseases), several of the ichthyoses (keratinization or scaling disorders), xeroderma pigmentosum caused by defective DNA repair genes, and basal cell carcinomas (the most common form of human cancer). In another potential application, genetically modified keratinocytes could be used for the systemic delivery of various cytokines, enzymes, and growth factors, such as human insulin and growth hormone. Finally, genes expressed in skin can effectively immunize animals against encoded antigens, such as tumor-associated or infectious organism-associated antigens, in a process known as DNA immunization or genetic vaccination. In the past, our laboratory has successfully introduced and expressed genes in the skin transiently, and demonstrated this approach is effective at DNA vaccination against Leishmaniasis. However, achieving long-term expression of desired genes in a significant percentage of keratinocytes has proved to be very difficult. Since unique cell surface markers for keratinocyte stem cells (KSC) are not yet known, the isolation, purification, and efficient introduction of genes into KSC that will be necessary for long-term expression in high percentages of keratinocytes is not yet possible. To address this issue, we are pursuing three complimentary approaches. First, in order to achieve long-term expression, we are using topical colchicine treatments to select for keratinocytes that express a multi-drug resistance (MDR) selectable marker gene along with a linked gene of interest. A significant advantage of this approach is that it does not depend on keratinocyte stem cell identification and targeting. When the selective treatment (colchicine) is applied topically, only keratinocytes that express the MDR selectable marker gene will survive and populate the epidermis. We have recently demonstrated the feasibility of this approach in an in vivo mouse model. When grafted keratinocytes that express MDR are topically selected with colchicine, long-term expression of MDR is maintained in a significant percentage of keratinocytes implying that KSC expressing MDR have been transduced and selected. Future studies linking the expression of MDR to other desired genes bicistronically will assess the therapeutic utility of this approach. Second, we would like to identify or develop viral vectors that are superior to retroviral vectors for gene delivery to KSC. Lentiviral vectors are of particular interest because they are superior in transducing and integrating into cells that are non-dividing or slowly dividing, and they may be superior for introducing genes into slowly cycling KSC. Somewhat surprisingly, the first series of experiments with our in vivo grafting assay to measure KSC transduction did not demonstrate that lentiviral vectors were superior to retroviral vectors. These studies suggest that retroviral vectors are able to transduce keratinocyte progenitor cells at an efficiency comparable to lentiviral vectors during ex vivo culture. Third, we have initiated studies to identify cell surface markers that are unique to KSC in order to distinguish KSC from other basal keratinocytes for efficient gene targeting. Keratinocyte stem cells are best identified by their ability to retain a BrdU label, and we have successfully FACS sorted pure populations of these "label retaining" cells or LRC. Alternatively, laser capture microdissection can be used to isolate the label-retaining keratinocyte stem cells. Finally, we have begun to investigate how a POU transcription factor, that is specifically expressed in epidermis, might influence and regulate epidermal differentiation. In vitro models of epidermal differentiation have been developed in which keratinocytes are transduced with retroviral vectors containing the POU transcription factor, and transgenic models have been made in which the POU transcription factor is either misexpressed in abnormal locations of the epidermis or mutated POU genes are expressed at the appropriate epidermal layers. Collaborators: Mark Udey, M.D., Ph.D.; Dermatology Branch, NCI Kim Yancey, M.D., Dermatology Branch, NCI Lorne Taichman, M.D.; Stony Brook, SUNY Paul Khavari, M.D., Ph.D.; Stanford Bill Telford, Ph.D. Medicine Branch, NCI